Influenza A viruses are divided into subtypes on the basis of the antigenicity of their surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA); influenza viruses bearing 15 HA and 9 NA subtypes have been isolated from birds, but only H1N1, H2N2, and H3N2 subtype viruses have circulated widely and caused epidemic disease in humans in the last century. Aquatic birds serve as a reservoir from which new subtypes of influenza A viruses enter the human population. In the last 10 years, human infections with avian influenza viruses (AIV) of three subtypes, H7, H5 and H9, have been detected on six occasions. There are several potential strategies for the development of vaccines to protect humans against influenza viruses, including (1) formalin inactivated whole or split virus, (2) HA subunit, and (3) live attenuated vaccines. Live attenuated vaccines generally induce broadly cross-reactive protection, which may be a useful feature in the event of a pandemic if a vaccine generated from the actual pandemic strain is not available. The cold-adapted (ca) influenza virus A/Ann Arbor/6/60 (AA) (H2N2) has been developed as a live attenuated vaccine seed virus that exhibits cold-adaptation, temperature-sensitive (ts), and attenuation (att) phenotypes which are specified by mutations in the internal genes. Reassortant H1N1 and H3N2 human influenza A viruses with the six internal gene segments of the AA ca virus have been repeatedly demonstrated to bear these phenotypes and extensive evaluation in humans has proven them to be attenuated and safe as live virus vaccines. This approach has recently been licensed for general use for interpandemic influenza A and B vius infections. We sought to use this strategy to develop vaccines that will be useful to protect against pandemic influenza A virus infection beginning with an H9N2 avian virus as model pandemic influenza A virus. Using classical genetic reassortment, we generated a reassortant virus (G9/AA ca) that contains the hemagglutinin and neuraminidase genes from influenza A/chicken/Hong Kong/G9/97 (H9N2) (G9) and six internal gene segments from the AA ca virus. The reassortant virus expressed ca and ts phenotypes and did not exhibit a high pathogenicity phenotype in chickens. When administered intranasally, the reassortant virus was immunogenic and protected mice from subsequent challenge with wild-type H9N2 viruses, although it was restricted in replication in the respiratory tract of mice. The G9/AA ca virus bears properties that are desirable in a vaccine for humans and is available for clinical evaluation and use, should the need arise. The data from this study provide further evidence that the internal protein genes of the AA ca virus specify these phenotypes irrespective of the subtype of the accompanying HA and NA genes. A single dose of G9/AA ca administered i.n. as a live virus vaccine was immunogenic and protected mice against subsequent challenge with homologous and antigenically heterologous H9N2 wt viruses. The in vitro and in vivo phenotypes and the safety profile in chickens exhibited by the G9/AA ca reassortant virus make this virus a suitable candidate pandemic influenza vaccine for evaluation in humans. Clinical trials are planned to establish the safety, infectivity, and immunogenicity of this vaccine in healthy adults. An optimal public health response in the event of a potential pandemic requires that vaccines be available to prevent infection with minimum delay and an important approach to pandemic preparedness is to generate and evaluate candidate vaccines against influenza A subtypes that are recognized to have pandemic potential, prior to their actual spread. The generation and evaluation of an H9N2 vaccine is a model of this approach. Vaccines against H5N2 subtype influenza are being generated; this reassortant virus will be evaluated in a similar fashion.